The present project aims at an analysis of feto-maternal interactions in the immune system mediated by the trophoblast-specific Class I gene product, HLA-G. The assembly and intracellular transport pathways traveled by HLA-G will be examined in trophoblast cells of human origin. The availability of HLA-G transgenic mice, together with mice transgenic for HLA-A2/human beta2m or HLA-B27/human beta2m, as well as mice deficient for mouse beta2m, will allow a detailed characterization of the dependency of HLA-G on the presence of beta2m in vivo. The dependency of HLA-G expression on peptides delivered by the MHC-encoded peptide transporter TAP will be examined by measuring TAP-dependent peptide transport in vitro in human trophoblast cells, and by examining the expression of HLA-G in trophoblast of mice transgenic for HLA-G, crossed onto a homozygous TAP-1 deficient background. To facilitate immunohistochemical and biochemical analysis, antibodies against unfolded, bacterially produced HLA-G will be generated, in a manner analogous to that used for the production of anti HLA-A,B,C heavy chain MoAbs. The availability of polyclonal anti-mouse Class I heavy chain sera will allow a search for a mouse equivalent of HLA-G, using procedures that led to the identification of HLA-G in man. The biochemical characterization of a mouse equivalent for HLA-G would constitute a first step towards the identification of the Class I gene that encodes it. Expression of HLA-G in cultured human trophoblast will be examined both under normal circumstances and in cells infected with Herpes Simplex Virus (HSV) or Cytomegalovirus (CMV). Each of these viruses down-modulates Class I expression in a unique manner, and has been associated with spontaneous abortion. If HLA-G protects the fetus from attack by the maternal immune system, then its down-regulation, as caused by viral infection, might have disastrous consequences for the fetus. The proposed work is relevant for understanding the molecular basis of feto-maternal immune interactions, and for understanding how viral pathogens impinge on them.
